Identification of therapeutic compounds

Abstract

Methods for identifying potential therapeutic agents involve determining the affinity and/or efficacy of a test compound for an adensoine receptor at a relatively high pH and at a relatively low pH. Compounds with greater affinity and/or efficacy at the low pH are identified as potential therapeutic agents, in particular for the treatment of pain or inflammation.

1 . A method for identifying a potential therapeutic agent, which comprises determining the affinity of a test compound for an adenosine receptor at a higher pH of at least 7.4, and also at a lower pH from 5.5 to 7.2, and identifying the compound as a said agent if the affinity at the lower pH is over 10 times greater than the affinity at the higher pH.  
     
     
         2 . A method according to  claim 1  wherein determining the affinity of the test compound comprises measuring binding of different concentrations of labelled test compound to the receptor.  
     
     
         3 . A method according to  claim 1  wherein determining the affinity of the test compound comprises measuring displacement of a bound labelled compound from the adenosine receptor with unlabelled test compound.  
     
     
         4 . A method according to  claim 1 , wherein the adenosine receptor is in a tissue, a tissue slice, an intact cell, a disrupted cell, or a cell derived membrane.  
     
     
         5 . A method for identifying a potential therapeutic agent, which comprises determining the efficacy of a test compound at an adenosine receptor at a higher pH of at least 7.4, and also at a lower pH from 5.5 to 7.2, and identifying the compound as a said agent if the efficacy at the lower pH is over 10 times greater than the efficacy at the higher pH.  
     
     
         6 . A method according to  claim 5 , wherein determining the efficacy of the test compound comprises contacting the test compound with a tissue, tissue slice, intact cell or partially disrupted cell comprising the adenosine receptor, and determining adenosine receptor action by measuring accumulation or depletion of a signalling molecule in the tissue, tissue slice, intact cell or partially disrupted cell.  
     
     
         7 . A method according to  claim 6  wherein the signalling molecule is cAMP, IP3 or free calcium.  
     
     
         8 . A method according to  claim 5 , wherein determining the efficacy of the test compound comprises contacting the test compound with a biological membrane comprising the adenosine receptor, and determining adenosine receptor action by measuring: activation of a G protein; activity of an enzyme; or ion flow through an ion channel associated with the biological membrane.  
     
     
         9 . A method according to  claim 8 , wherein activation of the G protein is measured by use of a radiolabelled guanine nucleotide, or wherein the enzyme activity measured is adenylyl cyclase, phosphodiesterase, phospholipase or protein kinase activity, or wherein the ion channel through which ion flow is measured is a calcium or a potassium channel.  
     
     
         10 . A method according to  claim 5 , wherein determining the efficacy of the test compound comprises contacting the test compound with a tissue, tissue slice, intact cell, disrupted cell or cell derived membrane comprising the adenosine receptor, and determining adenosine receptor action by measuring protein kinase activity in the tissue, tissue slice, intact cell, disrupted cell or cell derived membrane.  
     
     
         11 . A method according to  claim 5 , wherein determining the efficacy of the test compound comprises contacting the test compound with a tissue, tissue slice, intact cell, disrupted cell or cell derived membrane comprising the adenosine receptor, and determining adenosine receptor action by measuring phospholipase activity (e.g. phospholipase C, phospholipase A2, phospholipase D) in the tissue, tissue slice, intact cell, disrupted cell or cell derived membrane.  
     
     
         12 . A method according to  claim 5 , wherein determining the efficacy of the test compound comprises contacting the test compound with a tissue, tissue slice, intact cell, disrupted cell or cell derived membrane comprising the adenosine receptor, and determining adenosine receptor action by measuring protein phosphorylation and/or dephosphorylation in the tissue, tissue slice, intact cell, disrupted cell or cell derived membrane.  
     
     
         13 . A method according to  claim 1 , wherein the lower pH is from 5.5 to 6.5.  
     
     
         14 . A method according to  claim 1 , wherein the lower pH is from 5.5 to 6.2.  
     
     
         15 . A method according to  claim 1 , wherein the lower pH is from 5.0 to 7.0 rather than 5.5 to 7.2.  
     
     
         16 . A method according to  claim 1 , wherein the adenosine receptor is an adenosine A2A receptor, an adenosine A1 receptor, or an adenosine A3 receptor.  
     
     
         17 . A method according to  claim 1 , which further comprises determining whether the identified agent has a therapeutic effect.  
     
     
         18 . A method according to  claim 17  in which a non human animal model is used to determine whether the identified agent has a therapeutic effect.  
     
     
         19 . A method according to  claim 17  or  18 , wherein it is determined whether the identified agent has a therapeutic effect against pain or inflammation.  
     
     
         20 . A method according to  claim 17 , wherein it is determined whether the identified agent has a therapeutic effect against cancer, ischemia-reperfusion injury, excessive neuronal activity (for example in epilepsy, or chronic pain or hyperlagesia, including inflammatory and neuropathic pain), sepsis, septic shock, neurodegeneration (including Alzheimer's Disease), muscle fatigue or muscle cramp.  
     
     
         21 . A method according to  claim 17 , wherein it is determined whether the identified agent has a therapeutic effect against arthritis, asthma, psoriasis, bowel inflammation, rheumatoid arthritis, irritable bowel syndrome or osteoarthritis.  
     
     
         22 . A method according to  claim 17 , wherein it is determined whether the identified agent has a therapeutic effect against hyperalgesia caused as a result of neuropathy, including bowel pain, back pain, cancer pain, HIV pain, phantom limb pain, post-operative pain, diabetic neuropathy, polyneuropathy, post-herpes neuralgia, or trigeminal neuralgia.  
     
     
         23 . A method according to  claim 17 , wherein it is determined whether the identified agent has a therapeutic effect against hyperalgesia caused as a result of inflammatory disease, including bowel pain, back pain, cancer pain, fibromyalgia, post-operative pain, osteoarthritis, or rheumatoid arthritis.  
     
     
         24 . A method according to  claim 17  in which the therapeutic effect of the identified agent is determined at a concentration below the EC50 value, preferably one ten thousandth to one fifth of the EC50 value, of the agent at the adenosine receptor at pH 7.4.  
     
     
         25 . A method according to  claim 17  in which the side effects caused by the identified agent are determined.  
     
     
         26 . A method according to  claim 25  in which the side effects are determined at a concentration below the EC50 value, preferably one ten thousandth to one fifth of the EC50 value, of the agent at the adenosine receptor at pH 7.4.  
     
     
         27 . A method according to  claim 25  in which the side effects are bradycardia, hypotension or tachycardia side effects.  
     
     
         28 . A method for identifying a potential therapeutic agent which comprises contacting a test compound with an adenosine receptor in a pathological tissue, cell, or membrane and a corresponding normal tissue, cell, or membrane at a concentration below the EC50 value of the test compound at the adenosine receptor at pH 7.4, and determining whether there is any difference in action of the adenosine receptor in response to contact with the test compound between the normal tissue and the pathological tissue.  
     
     
         29 . A method according to  claim 28 , wherein the test compound is identified as a potential therapeutic agent if the test compound is an agonist of the adenosine receptor, and the action of the adenosine receptor in response to the test compound is greater in the pathological tissue than the normal tissue.  
     
     
         30 . A method according to  claim 28 , wherein the pathological tissue comprises epithelial tissue.  
     
     
         31 . A method according to  claim 30  for identifying a potential therapeutic agent for treating psoriasis, asthma, or COPD.  
     
     
         32 . A potential therapeutic agent identified by a method according to  claim 28 .  
     
     
         33 . A potential therapeutic agent according to  claim 32  for use as a medicament.